Note: Descriptions are shown in the official language in which they were submitted.
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Combretastatin A-1 Phosphate And Combretastatin B-1 Phosphate Prodrugs
Introduction
The present invention relates generally to the syntheses and structural
elucidation of Combretastatin A-1 Phosphate Prodrug and Combretastatin B-1
Phosphate Prodrug, and to the treatment of neoplastic diseases therewith.
This research was funded in part by Outstanding Investigator Grant
CA4434-05-10 awarded by the National Cancer Institute, DHHS. The United
States government may have certain rights to this invention.
Background Of The Invention
In 1987, the isolation and synthesis of combretastatin A-1 (1) and B-1
(2) from the South African willow tree Combretum caffrum (Combretaceae)
was reported (Pettit et al, 1987). Both natural products were shown to be
significant cancer cell growth inhibitors and antimitotic agents, providing an
EDso value of 0.99 tg/ml and 1.7 gg/ml respectively against the murine P388
lymphocytic leukemia in vitro system, and inhibiting microtubule assembly in
vitro with ICso values of 2 M (1) and 3 11M (11) (Pettit et al, 1987). A
comparison of diphenol 1 to the monophenol counterpart combretastatin A-4
(3a), the most active anticancer member of the combretastatin family, revealed
a very similar antimitotic activity (ICso 2-3 gM), but much greater
cytotoxicity
(EDso -0.0009 gg/ml, P388 cell line) (3a) (Pettit et al, 1989). However in
early murine (P388 leukemia) in vivo experiments, combretastatin A-1 led to
more consistent antineoplastic results (Pettit et al, 1987). The relevant
structures are shown inFigure 1, below.
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H3CO H3CO
H3CO \ / I OH H3CO
H3CO OH H3CO OR
OCH3 OCH3
3a, R=H, Combretastatin A-4
2, Combretastatin B-1 3b, R=P(O)(OCH C6H5)2
3c, R=P(O)(O7Na')2
Figure 1.
Development of combretastatin A-4 to the current Phase I human
cancer clinical trials was accelerated following synthesis of the phosphate
prodrug 3c from dibenzyl phosphate 3b and then uncovering its very promising
cancer antiangiogenesis effects. The phosphate derivative was chosen due to
the nature of its biolability and enhanced solubility characteristics. Once
administered, the phosphate prodrug is presumed to be converted into the
parent drug via non-specific phosphatases and then transported
intracellularly.
Phosphate 3c showed similar cytotoxicity when compared to the parent
compound (GIso 0.0004 gg/ml, P388 cell line), while greatly increasing the
aqueous solubility to 20 mg/ml. Prodrug 3c was also shown to induce vascular
shutdown within murine metastatic tumors at doses less than one-tenth of the
maximum tolerated dose.
The preclinical development of combretastatin A-1 (1) was hampered
owing to instability (oxidation to the 1,2-quinone) (Sackett, 1993; Haines,
1988) of the 2,3-dihydroxy unit. This was supported by the fact that
acetylation of 1 significantly enhanced cytotoxicity 10-fold, while reducing
the
inhibition of the tubulin assembly.
The synthesis of the combretastatin A-1 and B-1 phosphate prodrugs
were undertaken in order to improve solubility for drug delivery and to
increase
stability. Thus, the present invention is directed to the syntheses of
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combretastatin A-1 prodrug (ED50 <0.0100 p.g/ml, P388 cell line, 4),
combretastatin B-1 prodrug (ED50 0.335 p.g/ml, P388 cell line, 5), as shown in
Scheme 1, below, and a series of metal cation and ammonium cation salts of
the diphosphoric acid precursor 4 to evaluate effects on in vitro human cancer
cell growth and solubility behavior.
Scheme 1
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H3CO , I \
H3CO OH
H3CO OH
OCH3
1, Combretastatin A-1
(BnO)2P(O)H
CH3CN, CCI4, DMAP
DIPEA, -20 C
r-0
H3CO 0, ,O
P-0 \
H3CO 00
I /
n
H3CO O'PO /
~O \
OCH3
() TMSCI, Nal, CH3CN (i) H2, 10% Pd/C, CH3OH
(i) NaOCH3, CH3OH (ii) NaOCH3, CH3OH
p 3C p
H3CO 0 O 4Na+ H3CO \ I 0 O 4Na+ 11 H3CO WP~-O' H3CO 0.?.0-
OCH3 O OCH3 0-
5
4
Brief Summary Of The Invention
The original synthesis of combretastatin A-1 (1) was greatly improved
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to allow an efficient scale-up procedure for obtaining the antineoplastic
stilbene. Subsequent conversion to a useful prodrug was accomplished by
diphosphorylation (to 10) with in situ formation of dibenzylchlorophosphite
followed by cleavage of the benzyl ester protecting groups with
trimethyliodosilane. The phosphoric acid intermediate was treated with sodium
methoxide to complete a practical route to the sodium phosphate prodrug (4).
Selective hydrogenation of phosphate 10 and treatment of the product with
sodium methoxide led to combretastatin B-1 phosphate prodrug (5). The
phosphoric acid precursor of prodrug 4 was employed in a parallel series of
reactions to produce a selection of metal and ammonium cation prodrug
candidates. Each of the phosphate salts was evaluated from the perspective of
relative solubility behavior and cancer cell growth inhibition. The sodium
phosphate prodrug (4) of combretastatin A-1 was selected for detailed
antineoplastic studies.
Accordingly, the primary object of the present invention is the
discovery of phosphate prodrugs, which have been shown to exhibit greatly
improved properties in vitro and are designated herein as the combretastatin A-
1 phosphate prodrugs and combretastatin B-1 phosphate prodrug.
Another object of the present invention is the synthesis of metal and
ammonium cation derivatives of combretastatin A- 121,3 1-0-diphosphate
through the appropriate acid-base reaction.
These and still further objects as shall hereinafter appear are readily
fulfilled by the present invention in a remarkably unexpected manner as will
be
readily discerned from the following detailed description of an exemplary
embodiment thereof.
Description of the Preferred Embodiment
Materials and Methods
Ether refers to diethyl ether. All solvents were redistilled. Boron
trichloride (1.0 M solution in CH2C12), triphenylphosphine,
tetrabutylammonium fluoride (1.0 M solution in THF), dibenzyl phosphite,
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diisopropylethylamine (99%), chlorotrimethylsilane (99%), 4-
dimethylaminopyridine (DMAP), carbon tetrachloride (99%), zinc acetate
dihydrate, papavarine, and cesium hydroxide were obtained from Sigma-
Aldrich Chemical Company (Milwaukee, WI). Magnesium acetate
tetrahydrate, calcium acetate, manganese acetate, quinidine, quinine, and
concentrated hydrochloric acid were obtained from the Baker Chemical
Company. Verapamil and nicotinamide were purchased from the Alexis
Corporation. All other reagents were purchased from Acros Organics (Fisher
Scientific, Pittsburgh, PA).
Reactions were monitored by thin-layer chromatography using Analtech
silica gel GHLF Uniplates visualized under long-wave and short-wave UV
irradiation. Solvent extracts of aqueous solutions were dried over anhydrous
sodium sulfate. Where appropriate, the crude products were separated by
column chromatography, flash (230-400 Mesh ASTM) or gravity (70-230
Mesh ASTM) silica from E. Merck.
Melting points were measured with an electrothermal digital melting
point apparatus (model IA9200) and are uncorrected. The IR spectra were
obtained using a Mattson Instruments 2020 Galaxy Series FT-IR. ELMS data
were recorded with a MAT 312 mass spectrometer, and high-resolution FAB
spectra were obtained with a Kratos MS-50 mass spectrometer (Midwest
Center for Mass Spectrometry, University of Nebraska, Lincoln, NE). TOFMS
data were recorded with a Vestec Lasertec Research mass spectrometer
incorporating a Laser Sciences nitrogen laser that provided 337 nm light
pulses
of 3 ns duration with 4-hydroxybenzylidenemalononitrile as the matrix and
cytochrome c as the external standard for calibration purposes. Optical
rotation values were recorded employing a Perkin Elmer 241 polarimeter. The
UV spectra were recorded using a Hitachi U-2000 UV/VIS spectro-
photometer. All 'H and 13C NMR spectra were obtained using a Varian Gemini
300 MHz instrument with CDC13 (TMS internal reference) as solvent unless
otherwise noted. The 31P NMR spectra were obtained in CDC13, or D20 with
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85% H3PO4 as an external standard employing a Unity 500 MHz instrument.
Elemental analyses were determined by Galbraith Laboratories, Inc., Knoxville,
TN.
Upon initiation of this investigation directed at obtaining a useful
prodrug of combretastatin A-1 (1), synthesis (Pettit et al, 1987) of the
parent
compound required modification for a suitable scale-up procedure. Three
major improvements were needed: a more economic synthesis of 2,3-
dihydroxy-4-methoxy-benzaldehyde (6b); better separation of the bis-
(TBDMS) cis- and trans-isomers (8 and 9a) produced in the Wittig reaction;
and efficient desilyation of 8 to diphenol 1. A better route to aldehyde 6b
was
found to involve selective demethylation of 2,3,4-trimethoxybenzaldehyde (6a)
using a 1.0 M solution of boron trichloride in dichloromethane (Kaisalo et al,
1986). This method gave yields consistently in the 70% range, and the reaction
was conducted in a solvent that facilitated isolation of the water-soluble
diphenol (6b). The Wittig reaction sequence earlier used to afford stilbenes 8
and 9a relied on the separation by fractional recrystallization in ethanol.
Both
of these compounds were efficiently separated in the present study by column
chromatography (60:1:1, hexane:ethyl acetate:triethylamine). As expected, cis-
isomer 8 was easily converted to trans-isomer 9a by photoisomerization in high
yield (>80%) using 366 nm light (Waldeck, 1991; Pettit & Singh, 1987).
Finally, the desilylation of cis-isomer 8 to combretastatin A-i (1) using
tetrabutylammonium fluoride (TBAF) as originally described proved to be
unsatisfactory on a larger scale owing to formation of polymeric products.
However, diphenol 1, a base-sensitive catechol, was obtained in good yields
under acidic cleavage conditions employing 48% HBr (cat.) and potassium
fluoride in N,N-dimethylformamide (Sinhababu et al, 1988; Nelson & Crouch,
1996). Although the crude product produced from the original TBAF
desilylation procedure could be used directly in the phosphorylation step to
afford phosphate 10, it did not prove useful for obtaining pure combretastatin
A-1 (1) by column chromatography. trans-Stilbene 9a was also readily
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desilylated using either the 48% HBr (cat.)/KF or the TBAF method to afford
previously unreported diphenol 9b. The relevant structures are shown in
Figure 2, below.
Figure 2
O H P+(Ph)3Br
OR /
OR H3COOCH3
OCH3 OCH3
6a, R=CH3
6b, R=H 7
6c, R=Si(CH3)2C(CH3)3
H3CO / , OCH3
H3CO ' / OR H 3 A OR
OCH3 OR H3CO I OR
OCH3 OCH3
1, R=H 9a, R=Si(CH3)2C(CH3)3
8, R=Si(CH3)2C(CH3)3 9b, R=H
Once a practical scale-up synthesis of combretastatin A-1 (1) was in
hand, phosphorylation with dibenzyl phosphite (Silverberg et al, 1996) was
undertaken. Diphosphate 10 was obtained in high yield (97%). Removal of
the benzyl protecting groups was carried out with in situ generation of
trimethylsilyl iodide (TMSI) from reaction of sodium iodide and
chlorotrimethylsilane (Jung & Lyster, 1977; Olah et al, 1979; Morita et al,
1978; Jung & Lyster, 1977; Ho & Olah, 1976; Salomon et al, 1993). Initially,
TMSI-mediated cleavage afforded a large portion of the undesired trans-
isomer, presumably from the electrophilic addition of iodine to form an
iodonium ion and subsequent elimination to the trans-olefin (Hassner et al,
1970; Robertson et al, 1950; Zanger & Rabinowitz, 1975; Ayers et al, 1971;
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Skell & Pavlis, 1964). This problem was eventually circumvented through the
use of new sodium iodide and the correct dilution of acetonitrile needed for
the
debenzylation. At higher concentrations and with the use of older sodium
iodide the cleavage reaction produced a nearly 1:1 ratio of cis- to trans-
isomers. More dilute solutions and new sodium iodide led almost exclusively
to the desired cis- isomer (determined by NMR analysis).
The very successful benzyl ester cleavage reaction was preceeded by a
number of other approaches and reagents that proved to be in general
unsatisfactory. Unsuccessful debenzylation reactions applied to phosphate 10
included Raney nickel (W-2), ferric chloride, trimethylphenylthiosilane,
chromium trioxide, catalytic transfer hydrogenolysis, mild hydrogenation
(reaction times <10 min), DDQ, tripenylcarbenium, tin (IV) chloride, and
lithium hydroxide. In most instances the above reaction conditions resulted in
incomplete removal of the four benzyl groups and isomerization or reduction of
the olefin group. This debenzylation step proved to be the most challenging
synthetic obstacle in the synthesis of the desired prodrug, because of the
very
difficult isolation of the debenzylated diphosphate owing to its high
solubility in
water.
In order to try different protecting groups on the phosphate, several
other methods of phosphorylation were attempted. These included the use of
alkylamidophosphines, which have been shown to readily phosphorylate
alcohols and phenols in high yield. For example, di-tert-butyloxy(N,N-
diisopropylamido)phosphine, prepared from dichloro(N,N-
diisopropylamido)phosphine and tert-butanol was allowed to react with
diphenol 1 in the presence of IH-tetrazole. After phosphorylating diphenol 1,
subsequent in situ oxidation of the trivalent phosphorous to the pentavalent
species with meta-chloroperoxybenzoic acid did not result in the desired
phosphate. Perhaps this unpromising result arose from the steric crowding
involved with four tert-butyl groups in the 2'- and 3'- positions, or from the
oxidation step which may have effected the stilbene olefin. Interestingly, the
*Trade-mark
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use of dibenzyloxy(N,)V-diisopropylamido)phosphine under the reaction
conditions just described did afford phosphate 10 but only in 10% yield. Two
other phosphorylation methods were attempted using di-tert-butyl phosphite
with an in situ generation of the appropriate halide (Br, Cl), and N,N-
diisopropylphosphorodiamidic chloride. Neither method led to the
corresponding diphosphate analog of dibenzylphosphate 10.
Diphosphorylation of 1 was also achieved in good yield with diethylcyano-
phosphonate. However, this method proved to be problematic due to harsh
conditions needed to remove an ethyl group.
The combretastatin A-1 prodrug 4 was synthesized via the acid-base
reaction between the diphosphoric acid obtained from the sodium
iodide/chlorotrimethylsilane mediated debenzylation of 10, and sodium
methoxide in anhydrous methanol. Synthesis of the combretastatin B-1
prodrug (5) was carried out through the standard hydrogenation of diphosphate
10 followed by reaction with sodium methoxide in anhydrous methanol.
Bibenzyl prodrug 5 showed reduced antineoplastic activity when compared to
cis-stilbene prodrug 4, which is consistent with previous structure-activity
relationship studies in the combretastatin series. As expected, both exhibited
increased activity over their respective parent compounds. The relevant
structures are shown in Figure 3, below.
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H3C /
H3CO I OP03Z
H3CO OP03Z
OCH3
O 0
n
o O" Z+ -0 0-0 +
Z Z
11a, Z=Li+ 11h, Z=morpholine
11 b, Z=K+ 11 i, Z=piperazine
11c, Z=Cs+ 11j, Z=nicotinamide
11 d, Z=Mg 2+ Ilk, Z=quinine
2+ 111, Z=quinidine
11 e, Z=Ca 11m, Z=verapamil
11 f, Z=Mn2+ 11 n, Z=papaverine
11 g, Z=Zn2+
Figure 3. Metal cation and ammonium salts of combretastatin A-1 2',3'-O-d
!phosphate
Once an efficient method for the synthesis of prodrug 4 was in hand,
various metal cation and ammonium salts of the phosphoric acid precursor
were investigated. The cancer cell line and solubility behavior are summarized
in Table I, below. Of the monovalent metal cation salts, the lithium (11a, 40
mg/mL), sodium (4, 120 mg/mL), potassium (11b, >90 mg/mL) and cesium
(11c, >50 mg/mL) derivatives all showed good solubility in water while the
divalent metal cation salts derived from magnesium, calcium, manganese, and
zinc were progressively more insoluble owing perhaps to the formation of
polymers. The low solubility of these divalent metal cations did not allow for
a
suitable HRMS, LRMS, 'H or 13C NMR to be acquired. Of the ammonium
cation salts both the morpholine (11h, 50 mg/mL) and piperazine (11i, 34
mg/mL) showed excellent solubility characteristics. The remaining ammonium
cation salts showed solubility effects related to the corresponding amine. The
relevant structures of the amines and alkaloids used in Figure 3 are shown in
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Figure 4, below.
H H 0
CNl C;D fl"NH2
0 N
h, morpholine i, piperazine 1, nicotinamide
H H
HO H HO. H
H3CO / \ H3CO
IN \ IN
k, quinine 1, quinine
H3CO OCH3 OCH3
H3CO \ / \ H3CO
OCH3
H3CO i N
NC OCH3
m, verapamil n, papaverine
Figure 4. Amines and alkaloids used for the ammonium salts of combretastatin A-
1 2',3'-O-diphosphate
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The biological activities of the metal cation prodrugs 11a-g mainly
corresponded to the antimitotic properties of combretastatin A-1 (1) which
appears to be among the most potent antagonists of colchicine binding to
tubulin known, with nearly 99% inhibition of colchicine binding at equal
concentrations (Pettit et al, 1987; Lin et al, 1988; Sackett, 1993), and
thereby
inhibiting tubulin polymerization. Various cations also play a role in the
assembly or disassembly of microtubules. The relationship between site and
affinity is largely unclear; divalent cations interact with tubulin in complex
manners and are able to bind to them in both low- and high- affinity sites.
The
divalent cation magnesium has been shown to be essential for microtubule
assembly and has been proposed to bind as a complex with a nucleotide at the
exchangeable GTP [guanosine 5'-triphosphate ("GTP")] site. An adequate
concentration of potassium cations is required to facilitate the microtubule
formation and can be replaced by sodium ions. However, sodium ions begin to
suppress the polymerization at lower concentrations as compared to potassium
ions, suggesting that the factor involved is not the species of monovalent
cations, but rather the ionic strength. Calcium ions inhibit the
polymerization
of tubulin and upon addition to preassembled microtubules cause their
disassembly, while binding only weakly to tubulin at the magnesium site. A
manganous cation can be substituted for the magnesium cation with normal
microtubule assembly. Zinc cations have been shown to interfere with the
lateral binding between the protofilaments of microtubules. Metal cation
prodrugs 11a-g showed equal or less cytotoxicity versus the P388 and human
tumor cell lines when compared to prodrug 4 (Table I).
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Table I. Solubilities, Human Cancer Cell Line and Murine P-388 Lymphocytic
Leukemia Inhibitory Activities of Combretastatins A-1, A-4, B-1 and Synthetic
Modifications.
Solubility? Leukemia Pancreas-a Ovarian
Compound P388 BXPC-3 OVCAR-3
mg/ml ED50 g/ml
1 - 0.251 4.4 -
2 - 1.7 - -
3a - 0.0003 0.39 <0.001
3c 20 0.0004 - 0.023
4 120 <0.0100 1.5 0.024
>50 0.335 >10 2.0
ila 40 <0.0100 0.33 0.028
llb >90 0.0170 0.38 0.023
11c >50 0.0365 0.31 0.024
lld <1 0.0245 0.44 0.031
lie <1 0.0102 0.28 0.024
lif <1 0.715 4.7 0.044
llg <1 0.0394 0.36 0.027
llh 50 0.002 0.35 0.043
Ili 20 0.002 0.35 0.046
l i j 15 0.005 0.42 0.054
llk <1 0.004 0.47 0.054
111 <1 0.004 0.55 0.043
11m <1 0.004 0.60 0.066
lin <1 0.05 0.38 0.052
Compound Solubility? CNS Lung-NSC Colon Prostate
MG/M.. SF-295 NCI-H460 KM2OL2 DU-145
GI5o gg/ml
1 - - 0.74 - 0.17
2 - - - - -
3a - <0.001 0.0006 0.061 0.0008
3c 20 0.036 0.029 0.34 -
4 120 0.036 0.038 0.53 0.034
5 >50 2.3 3.3 >10 2.7
Ila 40 0.042 0.040 0.37 0.031
llb >90 0.035 0.036 0.30 0.024
11c >50 0.038 0.040 0.28 0.024
lld <1 0.039 0.039 0.47 0.032
lie <1 0.041 0.037 0.34 0.024
llf <1 0.28 0.19 6.1 2.4
llg <1 0.033 0.032 0.32 0.025
llh 50 0.042 0.046 0.26 0.039
Iii 20 0.039 0.037 0.20 0.036
lij 15 0.053 0.15 0.53 0.046
<1
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llk <1 0.044 0.34 0.40 0.050
ill <1 0.056 0.37 0.84 0.10
11m <1 0.070 0.40 1.2 0.086
lln 0.063 0.27 0.33 0.054
'Solubility values were obtained using lmL distilled water at 25 C
The ammonium cation prodrugs 11h-n were synthesized in order to
further evaluate aqueous solubility and to study their ability to reverse
multidrug resistance through interference with the P-glycoprotein mechanism
(11k-n) based on the amine in question. Morpholine, piperazine, and
nicotinamide (NADH biosynthesis) have had relatively limited clinical use. On
the other hand, cinchona alkaloids such as quinine and its sterioisomer
quinidine have been used to extensively treat malaria. Similarly, verapamil
has
been clinically shown to be a calcium antagonist, and a potent cardiovascular
agent with antianginal and antihypertensive properties, while also being used
in
the treatment of arrhythmias. Finally, papverine, isolated from opium, is best
known for its muscle relaxing properties. Ammonium cation prodrug llh-n all
showed strong antineoplastic properties equal to or better than their metal
cation counterparts (Table I).
2,3-Dihydroxy-4-methoxy-benzaldehyde (6b)
An anhydrous dichloromethane (500 mL) solution of 2,3,4-trimethoxy-
benzaldehyde (6a, 19.6 g, 100 mmol) under argon at ambient temperature was
stirred for 10 min and boron trichloride (100 mL, 100 mmol, 1 eq, 1.0 M soln
in dichloromethane) was added. After 2 hours, the second equivalent of boron
trichloride (100 mL, 100 mmol; 1 eq; 1.0 M solution in dichloromethane) was
added. The dark reaction mixture was stirred for 24 hours, and then slowly
poured into 10% sodium bicarbonate (aq) (40 g/360 mL). The resulting
solution was acidified with concentrated hydrochloric acid to pH 1. The
dichloromethane layer was separated, and the aqueous layer was extracted with
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ethyl acetate (4 x 100 mL) and dried. Evaporation of solvent in vacuo gave a
brown oil, which was absorbed onto silica gel and subjected to flash column
chromatography (50:50:1 hexane-ethyl acetate-acetic acid) to afford a yellow
solid. Recrystallization from ethyl acetate-hexane gave yellow needles (12.4
g;
74%): m.p. 115-116 C [lit. 116-117 C (Pettit et al, 1987)]; Rf 0.40 (1:1,
hexane-ethyl acetate); EIMS m/z 168 (100%, M+), 125 (25%), 122 (40%), 79
(20%), 52 (20%). Anal. Calcd. for CSHsO4: C, 57.14; H, 4.80. Found: C,
57.23; H, 4.79.
2,3-Bis-ftert-butyldimethylsilyloxyJ-4-methoxy-benzaldehyde (6c)
Preparation of silyl ether 6c was repeated essentially as originally described
(Pettit et al, 1987) from diphenol 6b (12.4 g) except for modification of its
purification procedure. Evaporation (under reduced pressure) of the ethyl
acetate used for extraction yielded a brown oil, which was absorbed onto
silica
gel and subjected to flash column chromatography (15:1 hexane-ethyl acetate).
The light yellow oily product was crystallized from methanol to afford the
title
compound as a colorless solid (25.5 g; 87% yield): m.p. 74-75 C [lit. 74.5-76
C (Pettit et al, 1987)]; Rf 0.80 (15:1, hexane-ethyl acetate); ELMS m/z 396
(2%, M+), 381 (10%), 339 (100%), 267 (15%), 73 (85%); 'H NMR (300
MHz, CDC13) S 0.14 (12H, s, 4 x SiCH3), 0.99 (9H, s, 3 x CH3), 1.05 (9H, s, 3
x CH3), 3.84 (3H, s, OCR), 6.63 (1H, d, J= 8.7 Hz, H-5), 7.49 (1H, d, J=
8.1 Hz, H-6), 10.23 (1H, s, CHO). Anal. Calcd. for C30IL6O4Si2: C, 60.56; H,
9.15. Found: C, 60.53; H, 9.38.
3,4,5-Trimethoxybenzyltriphenylphosphonium bromide (7)
Several modifications of the prior synthesis of this phosphonium bromide (7)
were employed here. The reaction mixture prepared from triphenylphosphine
(24.1 g, 92.0 mmol, 1.1 eq) and 3,4,5-trimethoxybenzyl bromide (21.3 g, 80.0
mmol) in toluene (200 mL) was heated at reflux for 6 hours and stirred for 18
hours at room temperature. Evaporation of the solvent in vacuo resulted in a
crude solid, which was recrystallized from ethyl acetate-hexane to afford
colorless crystals (39.0 g, 93%): m.p. 219-220 C [lit. 222-223 C (Pettit et
al,
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1987)]; Rf 0.00 (1:1, hexane-ethyl acetate); TOFMS m/z 443 [M-Br]+; IR
(film) v 1590, 1508, 1465, 1435, 1332, 1238, 1126, 997, 974, 873 cm-'; 'H
NMR (300 MHz, CDCb) 5 3.51 (6H, s, 2 x OCH3), 3.77 (3H, s, OCRs), 5.40
(2H, d, J= 14 Hz, Ph-CH2), 6.48 (21, bs, H-2, H-6), 7.70 (15H, m, P(Ph)3).
Anal. Calcd. for C28H2SO3PBr=1/2 H2O: C, 63.17; H, 5.49. Found: C, 62.97;
H, 5.63.
2'..3 '-Di[tert-butyldinsethylsilyl-oxyJ-(Z) and (E)-combretastatin A-1 (8 and
9a)
Except for the purification procedure, the earlier synthesis was repeated
using
26.5 g of aldehyde 6c. Evaporation (in vacuo) of the ethyl acetate solution
used for extraction afforded a crude brown oil. Subjection to silica gel flash
column chromatography (2x; 9:1 hexane-ethyl acetate followed by 60:1:1
hexane-ethyl acetate-triethylanune) led to both the pure (Z)- and (E)-isomers:
cis-Silyl ether 8 (16.1 g, 43% yield, colorless crystals) from ethanol: m.p.
130-
131 C [lit. 117-118 C (Pettit et al, 1987)]; Rf 0.44 (9:1, hexane-ethyl
acetate); ELMS m/z 560 (55%, M+), 503 (20%), 488 (40%), 431 (20%), 73
(100%); IR (film) v.2955, 2858, 1579, 1498, 1462, 1313, 1246, 1128, 1105,
842 cm'; 'H NMR (300 MHz, CDCb) S 0.11 (6H, s, SiCH3 x 2), 0.19 (6H, s,
SiCH3 x 2), 1.00 (9H, s, 3 x CH3), 1.04 (9H, s, 3 x CHs), 3.68 (6H, s, 2 x
OCH3), 3.74 (3H, s, OCH3), 3.84 (3H, s, OCH3), 6.36 (11L d, J= 12 Hz, -
CH=CH-), 6.36 (1H, d, J = 9.3 Hz, H-5'), 6.59 (1H, d, J = 12 Hz, -CH=CH-),
6.62 (2H, s, H-2, H-6), 6.89 (1H, d, J= 8.1 Hz, H-6'). Anal. Calcd. for
C3oH48O6Si2: C, 64.24; H, 8.62. Found: C, 64.30; H, 8.83. trans-Silyl ether 9a
(5.0 g, 13% yield) was obtained as a colorless fluffy solid from ethanol: m.p.
139-140 C [lit. 139-140 C (Pettit et al, 1987)]; Rf 0.40 (9:1, hexane-ethyl
acetate); ELMS m/z 560 (55%, M), 503 (10%), 488 (25%), 431 (15%), 73
(1000/o); IR (film) v., 1581, 1494, 1460, 1442, 1309, 1240, 1128, 1101, 837,
783 cm'; 'H NMR (300 MHz, CDCb) S 0.11 (61L s, SiCH3 x 2), 0.13 (61L s,
SiCH3 x 2), 1.00 (9H, s, 3 x CH3), 1.09 (9H, s, 3 x CH3), 3.79 (3H, s, OCH3),
3.86 (3H, s, OCH3), 3.88 (61L s, 2 x OCH3), 6.56 (1H, d, J= 8.7 Hz, H-5'),
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6.72 (2H, s, H-2, H-6), 6.80 (1H, d, J = 16 Hz, -CH=CH-), 7.20 (1H, d, J =
9.0 Hz, H-6'), 7.31 (1H, d, J= 16 Hz, -CH=CH-). Anal. Calcd. for
C30H0O6Si2: C, 64.24; H, 8.62. Found: C, 64.19; H, 8.94.
Photochemical isomerization of (Z)-stilbene 8 to (E)-stilbene 9a
A solution of 8 (10.6 g; 18.9 mmol) in chloroform was irradiated directly from
below with long-wave length (366 nm) UV for 5 hours. The ultraviolet source
was a UV lamp used for visualizing TLC plates equipped with both short-wave
(254 nm) and long-wave (366 nm) lamps. The product was separated by silica
gel flash column chromatography (9:1, hexane-ethyl acetate). The resulting
colorless solid was recrystallized from ethyl alcohol to yield (8.6 g; 81 %)
the
trans-stilbene 9a as a colorless fluffy solid. The product was identical
(spectroscopically) to the specimen synthesized in the preceding experiment.
Combretastatin A-1 (1)
Method A. Potassium fluoride (0.22 g, 3.79 mmol, 4 eq) was added to a
solution of bis-silyl ether 8 (0.52 g, 0.929 mmol) dissolved in DMF (6 mL
under argon at ambient temperature). The mixture was stirred for 5 min and a
catalytic amount of 48% aq. HBr (11 pL, 0.20 mmol, 0.2 eq) was added. After
18 hours of stirring, the resulting mixture was poured over ice-cold 6N
hydrochloric acid (aq). Following extraction of the mixture with ethyl acetate
(3 x 25 mL), the combined extract was washed with saturated sodium chloride
(aq) and dried. Removal of solvent in vacuo gave a light brown oil, which was
separated by column chromatography (50:50:1 hexane-ethyl acetate-acetic
acid) to afford a clear oil. Crystallization from ethyl acetate-hexane yielded
colorless crystals (0.20 g, 68% yield): m.p. 117-118 C [lit. 114-115 C
(Pettit
el al, 1987)]; Rt 0.67 (50:50:1 hexane-ethyl acetate-acetic acid); EIMS m/z
332
(100%, M+), 317 (90%), 257 (7%), 166 (5%), 115 (8%); 1R (film) v. 3443,
1624, 1581, 1504, 1462, 1329, 1238, 1124, 1093, 1001 cm'; 'H NMR (300
MHz, CDCb) 5 3.67 (6H, s, 2 x OCH3), 3.83 (3H, s, OCH3), 3.86 (3H, s,
OCH3), 5.38 (2H, s, 2 x OH, 2',3' D20 exchanged), 6.39 (1H, d, J= 9.0 Hz, H-
5'), 6.53 (211, s, H-2, H-6), 6.53 (11L d, J= 12 Hz, -CH--CH-, 6.60 (1 H, d,
J=
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12 Hz, -CH=CH-, 6.77 (1 H, d, J = 8.7 Hz, H-6'); 13C NMR (125 MHz, CDC13)
5152.71,146.29,141.65,137.16,132.54,132.46,130.11,124.02,120.27,
117.77, 105.88, 102.85, 60.79, 56.10, 55.77. Anal. Calcd. for C1sH2O06: C,
65.05; H, 6.06. Found: C, 65.04; H, 6.28.
Method B. To a solution of bis-silyl ether 8 (2.0 g, 3.53 mmol) in anhydrous
THE (10 mL) was added tetrabutylammonium fluoride (1 min THF; 7.8 mL,
7.80 mmol, 2.2 eq). The mixture was stirred for 25 min. Ice-cold 6N
hydrochloric acid (aq) was added, and the mixture was extracted with ethyl
acetate (4 x 25 mL). The combined organic extracts were washed with
saturated sodium chloride (aq) (50 mL) and dried. Removal of the solvent
under reduced pressure yielded a dark brown oil (quantitative yield), which
was
dried in high vacuum and then immediately phosphorylated to provide bis
phosphate ester 10.
2,3'-Dihydroxy-3,4,4,5-tetramethoxy-(E)-stilbene (9b, trans-combretastatin
A-1)
The same desilylation procedure was performed on silyl ether 9b described in
Method B above for the desilylation of 8 to combretastatin A-1 (1). trans-
Stilbene 9b was isolated following column chromatography (50:50:1 hexane-
ethyl acetate-acetic acid) as a clear oil that crystallized from ethyl acetate-
hexane: colorless crystals (1.0 g, 70% yield); m.p. 48-50 C; Rr 0.65 (50:50:1
hexane-ethyl acetate-acetic acid); TOFMS m/z 332 [M]+; IR (film) vm.,s 3408,
2937, 1622, 1581, 1510, 1464, 1290, 1238, 1126, 1003 cm 1; 'H NMR (300
MHz, CDC13) S 3.86 (3H, s, OCH3), 3.89 (3H,s, OCH3), 3.91 (6H, s, 2 x
OCH3), 6.50 (1H, d, J= 8.7 Hz, H-5'), 6.74 (2H, s, H-2,
H-6), 7.05 (1 H, d, J = 16 Hz, -CH=CH-), 7.05 (Ili, d, J = 8.7 Hz, H-6'), 7.23
(1H, d, J= 16 Hz, -C=C-). 13C NMR (125 MHz, CDC13) S 153.34, 146.24,
142.15, 133.95, 132.32, 127.84, 122.82, 118.37, 117.72, 103.46, 103.09,
60.93, 56.11. Anal.Calcd. for C18H20O6: C, 65.05; H, 6.06. Found: C, 64.53;
H, 6.41.
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2',3 '-0.Di[bis-benzylphosphorylJ-combretastatin A-1 (10)
To a solution of diphenol 1 (5.6 g; 17.0 mmol) in acetonitrile (100 mL cooled
to -20 C) was added carbon tetrachloride (16 mL, 170 mmol, 10 eq). The
resulting solution was stirred for 10 min prior to adding DIPEA (12 mL, 71
mmol, 4.2 eq via syringe) and DMAP (0.42 g, 3.40 mmol, 0.2 eq).
Approximately 1 min later, the slow (dropwise) addition of dibenzyl phosphite
(1 mL, 49 mmol; 2.9 eq) was begun at such a rate that the stirred reaction
mixture temperature was kept below -20 C. After 45 min, 0.5M KH2PO4 (aq)
was added and the mixture allowed to warm to room temperature. An ethyl
acetate extract (4 x 50 mL) was washed with saturated sodium chloride (aq),
followed by water, and dried. Removal of solvent in vacuo yielded a yellow oil
that was further separated by flash column chromatography (3:2 hexane-ethyl
acetate) to afford 14 g (97%) of a golden oil: Rt 0.31 (1:1, hexane-ethyl
acetate); ELMS m/z 852 (20%, M'), 762 (5%), 484 (40%), 277 (10%), 91
(100%); IR (film) vmax 2941, 2839, 1579, 1502, 1454, 1282, 1126, 1012, 966,
738 cm'; 'H NMR (300 MHz, CDC13) S 3.62 (6H, s, 2 x OCH3), 3.77 (3H, s,
OCH3), 3.80 (3H, s, OCH3), 5.08 (4H, in, 2 x CH2-Ph), 5.17 (4H, m, 2 x CH2-
Ph), 6.46 (2H, s, H-2, H-6), 6.51 (1 H, d, J = 12 Hz, -CH=CH-), 6.64 (1 H, d,
J
= 12 Hz, -CH=CH-), 6.67 (1H, d, J= 8.7 Hz, H-5'), 7.00 (1H, d, J= 8.1 Hz,
H-6'), 7.25 (20H, m, 4 x C6H5); 13C NMR (125 MHz, CDC13) 8 152.60,
151.36, 141.13, 137.04, 135.71, 135.65, 135.46, 135.40, 132.81, 131.87,
131.49, 128.25, 128.14, 127.71. 127.57, 126.67, 124.37, 124.16, 109.16,
106.00, 69.76, 69.72, 69.55, 69.51, 60.60, 56.23, 55.72; 31P NMR (202 MHz,
CDC13) S -4.81 (J = 2.6 Hz), -4.92 (J = 2.8 Hz). Anal. Calcd. for C46H46O12P2:
C, 64.79; H, 5.44. Found: C, 64.65; H, 5.53.
2',3 '-O-Dijbis-benqlphospkorylj -combretastatin A-1 (10)
1H-Tetrazole (70 mg; 0.96 mmol; 6.5 eq) was added in one portion to a stirred
solution of diphenol 1 (50 mg; 0.15 mmol) and dibenzyl N,N-
diisopropylphosphoramidite (0.12 g; 0.34 mmol; 2.2 eq) in dry tetrahydrofuran
(1 mL) and stirred for 15 min at room temperature. The mixture was then
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cooled to -50 C, and a solution of 85% meta-chloroperoxybenzoic acid in
dichloromethane was rapidly added such that the reaction temperature was
kept below 0 C. After stirring for 5 min at room temperature, 10% aqueous
sodium thiosulfate was added and the mixture stirred further for 10 min. The
resulting mixture was then extracted with ethyl acetate (4 x 10 mL), washed
with 10% aqueous sodium thiosulfate, 0.5 M aqueous sodium hydroxide, and
dried. Evaporation of the solvent under reduced pressure gave a yellow oil
that
was then subjected to flash column chromatography (3:2, hexane-ethyl acetate)
to afford a clear oil (15 mg; 10%) spectroscopically identical to the product
generated from the dibenzyl phosphite phosphorylation above.
2 ,3'-&Di[bis-ethylphosphorylJ -combretastatin A-1
Combretastatin A-1 (0.10 g; 0.30 mmol) was dissolved in dry dichloromethane
(5 mL). The solution was then cooled to 0 C and then diethyl
cyanophosphonate (0.10 mL; 0.66 mmol; 2.2 eq). followed by triethylamine
(0.17 mL; 1.2 mmol; 4 eq) were added. After stirring at 0 C for 2.5 hours the
mixture was extracted with dichloromethane (4 x 20 mL), the combined
organic extract washed with water, and dried. Evaporation of the solvent in
vacuo yielded a light yellow oil which was purified by flash column
chromatography (3:2, ethyl acetate-hexane) to afford a clear oil (0.14 g;
75%):
Rf 0.38 (3:2, ethyl acetate-hexane); EIMS m/z 604 (100%, M), 468 (10%),
369 (5%), 206 (5%), 45 (20%); IR (film) v. 2984, 1608, 1579, 1504, 1454,
1419, 1327, 1273, 1240, 1126 cm';'HNMR(300MHz, CDC13) 81.31-1.39
(12 H, m, 4 x OCH2OCH3), 3.66 (6H, s, 2 x OCH3), 3.81 (3H, s, OCH3), 3.85
(3H, s, OCH3), 4.2-4.34 (8H, m, 4 x OCH2OCH3), 6.48 (2H, s, H-2, H-6),
6.56 (1 H, d, J = 12 Hz, -CH=CH-), 6.65 (1 H, d, J = 7.8 Hz, H-5'), 6.66 (1 H,
d, J= 12 Hz, -CH=CH-), 6.97 (1H, d, J= 8.7 Hz, H-6'). Anal. Calcd. For
C26H3sOi2P2: C, 51.66; H, 6.34. Found: C, 51.66; H, 6.46.
Sodium combretastatin A-12,3 '-O-&phosphate (4)
To a solution of phosphate 10 (3.2 g, 3.69 mmol) in acetonitrile (40 mL) under
argon was added sodium iodide (2.2 g, 14.8 mmol, 4 eq). Before dropwise
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addition of chlorotrimethylsilane (1.9 mL, 14.9 mmol, 4 eq), the mixture was
stirred for 2 min, and 30 min later the reaction was terminated with 1% aq
sodium thiosulfate (4 mL). Removal of the acetonitrile in vacuo afforded a
crude mixture, which was dissolved in water-dichloromethane and washed with
water (4 x 10 mL). Concentration (facilitated by toluene azeotrope) of the
aqueous layer resulted in isolation of the crude phosporic acid intermediate
which was subjected to drying in high vacuum (I hour) and then dissolved in
dry methanol (10 mL under argon). Next sodium methoxide (0.80 g, 14.8
mmol, 4 eq) was added. The mixture was allowed to stir (6 hours) and
additional methanol was added to effect dissolution. After filtration of the
solution, concentration of the methanol in vacuo led to an off-white solid,
which was reprecipitated from water-ethanol to yield a colorless powder (1.7
g,
81% yield): m.p. 168-170 C (dec.); UV Amax (H2O) 298 nm (log e, 4.16); IR
(KBr) v 3364, 1647, 1579, 1506, 1446, 1315, 1238, 1126, 1093, 991 cm 1;
1H NMR (300 MHz, D20) 6 3.63 (6H, s, 2 x OCH3), 3.66 (6H, s, 2 x OCH3),
6.41 (1 H, d, J = 12 Hz, -CH=CH-), 6.43 (1 H, d, J = 8.7 Hz, H-5'), 6.70 (2H,
s,
H-2, H-6), 6.81 (1H, d, J = 8.7 Hz, H-6'), 6.94 (1H, d, J = 12 Hz, -C=C-); 13C
NMR (75 MHz, CD30D) 6 153.56, 153.12, 136.73, 135.31, 129.12, 128.50,
124.88, 124.36, 107.71, 61.89, 57.16, 56.82; 31P NMR (202 MHz, D20) 6
2.07, 1.78. HRFAB MS m/z (peak height) 580.9958 (100%, M+H), Calcd. for
C 1 sH l9O 12Na4 P 2: 580.9942.
Sodium combretastatin B-12'Y3 '-O-diphosphate (5)
To a solution of phosphate 10 (1.1 g, 1.28 mmol) in methanol (5 mL in a
hydrogenation flask), was added 10% Pd/C (1.1 g, 1 wt. eq). The mixture was
hydrogenated for 24 hours at 35 psi. Filtration of the solution through celite
and subsequent evaporation of solvent in vacuo afforded a light brown oil.
Anhydrous methanol (5 mL) was added to the crude diphosphate followed by
sodium methoxide (0.28 g, 5.13 mmol, 4 eq). The mixture was stirred for 6
hours, at which point additional methanol was added until the product
dissolved. Filtration of the methanol solution and subsequent concentration in
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vacuo afforded a colorless solid, which was reprecipitated from methanol-
acetone to yield a colorless powder (0.55 g, 74% yield): m.p. 170-172 C
(dec.); UV 7 (H20) 269 nm (log F, 3.24); 1R (KBr) vmaa 3385, 1589, 1496,
1458, 1236, 1186, 1124, 1087, 995, 559, cni 1; 'H NMR (300 MHz, D20)8
2.75 (2H, t, J = 7.2 Hz, CH2), 2.94 (2H, t, J = 8.1 Hz, CH2), 3.62 (3H, s,
OCH3), 3.67 (3H, s, OCH3), 3.73 (6H, s, 2 x OCH3), 6.65 (1H, d, J= 8.1 Hz,
H-5'), 6.61 (2H, s, H-2, H-6), 6.66 (1 H, d, J = 9.0 Hz; H-6'), 13C NMR (100
MHz, CDC13)5 153.13, 152.20, 146.31, 141.01, 137.13, 135.55, 129.30,
124.00, 108.05, 107.28, 61.85, 57.08, 56.84, 36.88, 32.50; 31P NMR (162
MHz, D20) 8 1.74, 1.36. HRFAB MS m/z (peak height) 583.0097 (100%,
M+H), Calcd. for C18H2,0,2Na4P2: 583.0099.
General Procedure for Synthesis of the Combretastatin A-1 Phosphate
Prodrugs
Method A. Each of the metal cation-containing salts was obtained by this
procedure as outlined directly below f o r preparing the tetralithium salt h
a.
Lithium combretastatin A-1 213 '-0-diphosphate (11 a)
To a solution of phosphate 10 (0.42 g, 0.488 mmol) in acetonitrile (5 mL,
under argon) was added sodium iodide (0.29 g, 1.95 mmol, 4 eq). The mixture
was stirred for 2 min, and chlorotrimethylsilane (0.25 mL, 1.95 mmol, 4 eq)
was added (dropwise). After stirring for 30 min, the reaction was stopped with
1% aq sodium thiosulfate (2 mL). Removal of the acetonitrile in vacuo
afforded a residue that was treated with 1.0 M lithium hydroxide dissolved in
methanol (2.1 mL, 2.1 mmol, 4.1 eq) for 6 hours. The product was
reprecipitated from water-ethanol to yield an off white powder (0.23 g, 92%):
m.p. 138-140 C (dec.); IR (KBr) vmu 3311, 1579, 1508, 1442, 1303, 1240,
1167, 1132, 1012, 533, cm'; 'H NMR (300 MHz, D20) S 3.62 (6H, s, 2 x
OCH3), 3.65 (6H, s, 2 x OCH3), 6.42 (1H, d, J= 13 Hz, -CH=CH-), 6.43 (1 H,
d, J = 7.2 Hz, H-5'), 6.69 (2H, s, H-2, H-6), 6.80 (111, d, J = 8.7 Hz, H-6'),
6.91 (1H, d, J= 12 Hz, -CH=CH-); LRFAB MS: m/z (peak height) 509
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[(anion + 3 Li)-, 50%], 503 [(anion + 2 Li + H)-, 100%], 497 [(anion + Li +
2H)-, 80%].
Potassium combretastatin A-12,3 '-&-&Pkospkate (11 b)
The potassium salt reprecipitated from water-ethanol as a colorless powder
(0.27 g; 83%): m.p. 113-115 C (dec.); IR (KBr) v. 3383, 1653, 1579, 1506,
1456, 1419, 1126, 1089, 989, 545 cm'; 'H NMR (300 MHz, D20) S 3.62 (6H,
s, 2 x OCH3), 3.65 (3H, s, OCH3) 3.66(3H, s, OCH3), 6.42 (1H, d, J= 12Hz, -
CH=CH-), 6.44 (1H, d, J= 8.7 Hz, H-5'),6.68 (2H, s, H-2, H-6), 6.81 (1H, d,
J = 8.1 Hz, H-6'), 6.91 (1 H, d, J = 12 Hz, -CH=CH-).
Cesium combretastatin A-12 ,3 -"phosphate (11c)
Reprecipitation from water-ethanol yielded a colorless powder (0.22 g; 36%)
m.p. 142-144 C (dec.); IR (KBr) v. 3385, 1577, 1506, 1456, 1419, 1238,
1126, 1089, 985, 545 cm'; 'H NMR (300 MHz, D20) 8 3.61 (6H, s, 2 x
OCH3), 3.65 (3H, s, OCH3), 3.66 (3H, s, OCH3), 6.43 (1H, d,
J= 13 Hz,-CH=CH-), 6.45 (1H, d, J= 8.7 Hz, H-5'), 6.67 (2H, s, H-2,H-6),
6.80 (1H, d, J= 8.7 Hz, H-6'), 6.89 (1H, d, J= 13 Hz, -CH=CH-).
Magnesium combretastatin A-12,3 '-O-diphosphate (11 d)
The precipitate from the reaction was filtered and washed with water to afford
a cream colored powder (0.20 g; 80%) m.p. 150-152 C (dec.); IR (KBr) v.
3421, 1635, 1579, 1498, 1446, 1236, 1126, 1099, 1006, 547 cm'.
Calcium combretastatin A-12,3 -O-diphosphate (Ile)
The precipitate from the reaction was filtered and washed with water to afford
a cream-colored powder (0.24 g; 70%): m.p. 163-165 C (dec.); IR (KBr) v.
3445, 1577, 1506, 1456, 1238, 1126, 1097, 1004, 837, 526 cm'. LRFAB MS
(peak height) 529 [(anion + Ca + H)', 10%].
Manganese combretastatin A-12 ,3'-O-diphosphate (11j)
The precipitate from the reaction was filtered and washed with water to afford
a tan powder (0.12 g; 55%) m.p. 135-137 C (dec.); IR (KBr) v. 3447, 1575,
1506, 1456, 1317, 1126, 1095, 1004, 667, 518 cm'. LRFAB MS m/z (peak
height) 543 [(anion + Mn + H)', 15%].
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Dizinc combretastatin A-1 2 ;3'-O-diphosphate (11g)
The precipitate from the reaction was filtered and washed with water to afford
a colorless powder (0.28 g; 86%): m.p. 243-245 C (dec.); IR (KBr) vmõs 3441,
1579, 1506, 1456, 1421, 1315, 1238, 1163, 1126, 1097, cm'.
Method B. Each of the ammonium cation salts (llh-n) of combretastatin A-1
phosphate was prepared by this general procedure. The same method as
described for prodrugs lla-g was used, except that the appropriate amine or
alkaloid (4 eq.) was added to the phosphoric acid to yield prodrugs l lh-n.
All
reaction mixtures were stirred for 8 hours and
recrystallization/reprecipitation
was performed with methanol-ether unless otherwise stated. These ammonium
cation salts were investigated by HRFAB MS and the results were erratic.
Presumably, this was due to various anion cation combinations and other types
of associations. However, in each case it was clear that a salt of the same
composition was obtained that was suitable for our purposes.
Morpholine combretastatin A-12,3 '-OL-&Pkosphate (11h) Reprecipitation
yielded a colorless solid (0.26 g): m.p. 168-170 C (dec.); IR (KBr) v. 3402,
3014, 2868, 2470, 1579, 1498, 1450, 1313, 1126, 1103 cm'; 'H NMR (300
MHz, D20) ,S 3.12 (8H,t, J = 4.5 Hz, CH2OCH2x2), 3.61 (6H, s, 2xOCH.3),
3.66 (3H, s, OCH3), 3.72 (3H, s, OCH3), 3.81 (8H, t, J= 4.8
Hz,CH2NCH2x2), 6.51 (1H, d, J= 12 Hz, -CH=CH-), 6.55 (1H, d, J= 8.7 Hz,
H-5'), 6.61 (2H, s, H-2, H-6), 6.74 (1 H, d, J= 12 Hz, -CH=CH), 6.81 (1 H, d,
J = 8.7 Hz, H-6').
Piperatine combretastatin A-12,3'-O-diphosphate (Ili). Reprecipitation
from ethanol-water yielded a colorless solid (0.34 g): m.p. 139-141 C (dec.);
IR (KBr) v 3406, 3005, 2839, 1579, 1498, 1446, 1126, 1093, 989, 949 cm
'; 'H NMR (300 MHz, D20) S 3.05 (6H, brs, -CH2-), 3.59 (6H, s, 2xOCH3),
3.65 (3H, s, OCH3), 3.71 (3H, s, OCH3), 6.51 (1H, d, J=12 Hz, -CH=CH-),
6.54 (1H, d, J = 8.4 Hz, H-5'), 6.60 (2H, s, H-2, H-6), 6.72 (1H, d, J = 12
Hz,
-CH=CH-), 6.79 (1 H, d, J = 8.7 Hz, H-6').
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Nicotinamide combretastatin A-1 2,3 '-O-&phosphate (11j). Reprecipitation
yielded a cream-colored solid (0.46 g): m.p. 148-150 C (dec.); IR (KBr) v.
3350, 3090, 2937, 2837, 1689, 1577, 1498, 1448, 1124, 1097 cm'; 'H NMR
(300 MHz, D20) 3 3.54 (6H, s, 2 x OCH3), 3.60 (3H, s, OCI-3), 3.72 (3H, s,
OCH3), 6.46 (2H, s, H-2, H-6), 6.47 (1H, d,.[---12 Hz, -CH=CH-), 6.57 (1H, d,
J = 8.7 Hz, H-5'), 6.61 (1 H, d, J = 11 Hz, -CH=CH-), 6.75 (1 H, d, J = 8.7
Hz,
H-6'), 8.02 (1H, dd, J= 8.4 Hz), 8.77 (1H, d, J= 8.1 Hz), 8.82 (1H, d, J= 8.4
Hz), 9.05 (1H, s).
Quinine combretastatin A-12'..3 '-O-dephosphate (Ilk). Reprecipitation
yielded a cream-colored solid (0.48 g): m.p. 144-146 C (dec.); [a]n25 -35 (c
= 1.12, MeOH); IR (KBr) v. 3383, 2941, 1620, 1579, 1504, 1446, 1240,
1126, 1091, 987 cm'; 'H NMR (300 MHz, D20) 3 1.25 (2H, brs), 1.69 (3H,
brs), 1.87 (1H, brs), 2.49 (2H, brs), 2.93 (2H, brs), 3.20 (1H, brs), 3.50
(6H, s,
2 x OCH3), 3.57 (3H, s, OCH3), 3.62 (3H, s, OCH3), 3.80 (3H, s, OCH3),
4.74-5.10 (21L m) 5.48-5.60 (1H, m), 5.81 (1H, brs), 6.32 (1H, d, J= 12 Hz, -
CH=CH-), 6.37 (1 H, d, J = 9.0 Hz, H-5'), 6.43 (2H, s, H-2, H-6), 6.67 (1 H,
d,
J = 8.7 Hz, H-6'), 6.70 (1H, d, J = 12 Hz, -CH=CH-), 7.14 (1H, d, J = 11 Hz),
7.27 (1H, d, J = 7.5 Hz), 7.49 (1H, d, J = 4.5 Hz), 7.78 (114, d, J = 10 Hz),
8.51 (1H, d, J = 4.2 Hz).
Quinidine combretastatin A-12 ,3'-O-diphosphate (11l). Reprecipitation
yielded a light cream-colored solid (0.57) g: m.p. 158-160 C (dec.);
[a]D0+88 (c = 1.05, MeOH); IR (KBr) v. 3385, 3084, 2943, 2359, 1622,
1510, 1454, 1244, 1126, 1093 cm'; 'H NMR (300 MHz, CD3OD) 3 0.94 (2H,
m), 1.54-1.67 (3H, m), 1.75 (1H, brs), 2.14-2.21 (2H, m), 2.40 (2H, m), 2.95-
3.16 (1H, m), 3.49 (6H, s, 2xOCH3), 3.56 (3H, s, OCH3), 3.58 (3H, s, OCH3),
3.81 (311, s, OCH3), 4.99-5.07 (2H, m), 5.88-5.99 (1H, m), 6.04 (1H, brs),
6.23 (1 H, d, J= 13 Hz, -CH=CH-), 6.35 (1 H, d, J= 8.4 Hz, H-5'), 6.36 (211,
s,
-2, H-6), 6.68 (1 H, d, J = 8.4 H, H-6'), 6.76 (111, d, J = 13 Hz, -CH=CH-),
7.21-7.26 (2H,m), 7.58 (1H, d, J= 4.8 Hz), 7.78 (1H, d, J= 8.7 Hz),- 8.52
(1H,d,J=4.2Hz).
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Verapamil combretastatin A-12',3 '-O-&pkosphate (11m). Reprecipitation
yielded a light cream colored solid (0.39 g): m.p. 160-162 C.; IR (KBr) v.,
3427, 2960, 2362, 1577, 1498, 1452, 1217, 1126, 1060, 945 cm-1 ; IH NMR
(300 MHz, D20/CD3OD) 8 0.58 (3H, d, J= 6.6 Hz, (CH3)2CH), 1.00 (3H, d, J
= 6.6 Hz, (CH3)2CH),. 1.42-2.16 (5H, m, CH2CtCH2N and (CH3)2CH), 2.65
(3H, s, NCH3), 2.79-3.02 (6H, m, CH2 NCH2CH. PH), 3.51 (6H, s, 2 x OCH3),
3.58 (3H, s, OCH3), 3.65 (3H, s, OCH3), 3.66 (9H, s, 3 x OCH3), 3.67 (3H, s,
OCH3), 6.45 (1H, d, J= 12 Hz, -CH=CH-), 6.49 (2H, s, H-2, H-6), 6.53 (1H,
d, J= 8.7 Hz, H-5'), 6.65 (1 H, d, J= 12 Hz, -CH=CH-), 6.67-6.86 (61-1, m,
Aryl H's), 6.72 (1 H, d, J= 8.7 Hz, H-6').
Papaverine combretastatin A-12 ,3'-O-diphosphate (11n). Reprecipitation
yielded a cream colored solid (0.65 g): m.p. 149-151 C (dec.); IR (KBr) v.
3447, 2937, 2837, 2449, 1605, 1510, 1452, 1298, 1234, 1126 cm 1; 1H NMR
(300 MHz, D20/CD3OD) 8 3.44 (6H, s, 2 x OCH3), 3.54 (3H, s, OCH3), 3.61
(3H, s, OCH3) 3.62 (3H, s, OCH3), 3.63 (3H, s, OCH3), 3.78 (3H, s, OCH3),
3.89 (3H, s, OCH3), 4.50 (2H, s, -CH2-), 6.28 (1H, d, J= 12 Hz, -CH=CH-),
6.32 (2H, s, H-2, H-6), 6.42 (l H, d, J= 9.0 Hz, H-5'), 6.52 (1 H, d, J= 12
Hz,
-CH=CH-), 6.61 (111, d, J= 8.7 Hz, H-6'), 6.65 (1H, d, J= 9.3 Hz), 6.74 (1H,
d, J= 8.1 Hz), 6.85 (1H, s), 7.81 (1H, d, J= 6.6 Hz) 8.09 (111, s).
Dosages
The dosage administered will be dependent upon the identity of the
neoplastic disease; the type of host involved, including its age, health and
weight; the kind of concurrent treatment, if any; the frequency of treatment
and
therapeutic ratio.
Illustratively, dosage levels of the administered active ingredients are:
intravenous, 0.1 to about 200 mg/kg; intramuscular, 1 to about 500 mg/kg;
orally, 5 to about 1000 mg/kg; intranasal instillation, 5 to about 1000 mg/kg;
and aerosol, 5 to about 1000 mg/k of host body weight.
Expressed in terms of concentration, an active ingredient can be present
in the compositions of the present invention for localized use about the
cutis,
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WO 01/81355 PCT/US01/13858
intranasally, pharyngolaryngeally, bronchially, intravaginally, rectally, or
ocularly in concentration of from about 0.01 to about 50% w/w of the
composition; preferably about 1 to about 20% w/w of the composition; and for
parenteral use in a concentration of from about 0.05 to about 50% w/v of the
composition and preferably from about 5 to about 20% w/v.
The compositions of the present invention are preferably presented for
administration to humans and animals in unit dosage forms, such as tablets,
capsules, pills, powders, granules, suppositories, sterile parenteral
solutions or
suspensions, sterile non-parenteral solutions of suspensions, and oral
solutions
or suspensions and the like, containing suitable quantities of an active
ingredient.
For oral administration either solid or fluid unit dosage forms can be
prepared.
Powders are prepared quite simply by comminuting the active
ingredient to a suitably fine size and mixing with a similarly comminuted
diluent. The diluent can be an edible carbohydrate material such as lactose or
starch. Advantageously, a sweetening agent or sugar is present as well as a
flavoring oil.
Capsules are produced by preparing a powder mixture as hereinbefore
described and filling into formed gelatin sheaths. Advantageously, as an
adjuvant to the filling operation, a lubricant such as talc, magnesium
stearate,
calcium stearate and the like is added to the powder mixture before the
filling
operation.
Soft gelatin capsules are prepared by machine encapsulation of a slurry
of active ingredients with an acceptable vegetable oil, light liquid
petrolatum or
other inert oil or triglyceride.
Tablets are made by preparing a powder mixture, granulating or
slugging, adding a lubricant and pressing into tablets. The powder mixture is
prepared by mixing an active ingredient, suitably comminuted, with a diluent
or
base such as starch, lactose, kaolin, dicalcium phosphate and the like. The
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powder mixture can be granulated by wetting with a binder such as corn syrup,
gelatin solution, methylcellulose solution or acacia mucilage and forcing
through a screen. As an alternative to granulating, the powder mixture can be
slugged, i.e., run through the tablet machine and the resulting imperfectly
formed tablets broken into pieces (slugs). The slugs can be lubricated to
prevent sticking to the tablet-forming dies by means of the addition of
stearic
acid, a stearic salt, talc or mineral oil. The lubricated mixture is then
compressed into tablets.
Advantageously, the tablet can be provided with a protective coating
consisting of a sealing coat or enteric coat of shellac, a coating of sugar
and
methylcellulose and polish coating of carnauba wax.
Fluid unit dosage forms for oral administration such as in syrups, elixirs
and suspensions can be prepared wherein each teaspoonful of composition
contains a predetermined amount of an active ingredient for administration.
The water-soluble forms can be dissolved in an aqueous vehicle together with
sugar, flavoring agents and preservatives to form a syrup. An elixir is
prepared
by using a hydroalcoholic vehicle with suitable sweeteners together with a
flavoring agent. Suspensions can be prepared of the insoluble forms with a
suitable vehicle with the aid of a suspending agent such as acacia,
tragacanth,
methylcellulose and the like.
For parenteral administration, fluid unit dosage forms are prepared
utilizing an active ingredient and a sterile vehicle, water being preferred.
The
active ingredient, depending on the form and concentration used, can be either
suspended or dissolved in the vehicle. In preparing solutions the water-
soluble
active ingredient can be dissolved in water for injection and filter
sterilized
before filling into a suitable vial or ampule and sealing. Advantageously,
adjuvants such as a local anesthetic, preservative and buffering agents can be
dissolved in the vehicle. Parenteral suspensions are prepared in substantially
the same manner except that an active ingredient is suspended in the vehicle
instead of being dissolved and sterilization cannot be accomplished by
filtration.
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The active ingredient can be sterilized by exposure to ethylene oxide before
suspending in the sterile vehicle. Advantageously, a surfactant or wetting
agent
is included in the composition to facilitate uniform distribution of the
active
ingredient.
In addition to oral and parenteral administration, the rectal and vaginal
routes can be utilized. An active ingredient can be administered by means of a
suppository. A vehicle which has a melting point at about body temperature or
one that is readily soluble can be utilized. For example, cocoa butter and
various polyethylene glycols (Carbowaxes) can serve as the vehicle.
For intranasal instillation, a fluid unit dosage form is prepared utilizing
an active ingredient and a suitable pharmaceutical vehicle, preferably P.F.
water, a dry powder can be formulated when insufflation is the administration
of choice.
For use as aerosols, the active ingredients can be packaged in a
pressurized aerosol container together with a gaseous or liquefied propellant,
for example, dichlorodifluoromethane, carbon dioxide, nitrogen, propane, and
the like, with the usual adjuvants such as cosolvents and wetting agents, as
may
be necessary or desirable.
The term "unit dosage form" as used in the specification and claims
refers to physically discrete units suitable as unitary dosages for human and
animal subjects, each unit containing a predetermined quantity of active
material calculated to produce the desired therapeutic effect in association
with
the required pharmaceutical diluent, carrier or vehicle. The specifications
for
the novel unit dosage forms of this invention are dictated by and are directly
dependent on (a) the unique characteristics of the active material and the
particular therapeutic effect to be achieved, and (b) the limitation inherent
in
the art of compounding such an active material for therapeutic use in humans,
as disclosed in this specification, these being features of the present
invention.
Examples of suitable unit dosage forms in accord with this invention are
tablets, capsules, troches, suppositories, powder packets, wafers, cachets,
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teaspoonfuls, tablespoonfuls, dropperfuls, ampules, vials, segregated
multiples
of any of the foregoing, and other forms as herein described.
The active ingredients to be employed as antineoplastic agents can be
easily prepared in such unit dosage form with the employment of
pharmaceutical materials which themselves are available in the art and can be
prepared by established procedures. The following preparations are
illustrative
of the preparation of the unit dosage forms of the present invention, and not
as
a limitation thereof. Several dosage forms were prepared embodying the
present invention. They are shown in the following examples in which the
notation "active ingredient" signifies either phenstatin 3b and/or phenstatin
prodrug 3d, and/or benzophenones 4a-f or any other compound described
herein.
COMPOSITION "A"
Hard-Gelatin Capsules
One thousand two-piece hard gelatin capsules for oral use, each capsule
containing 200 mg of an active ingredient are prepared from the following
types and amounts of ingredients:
Active ingredient, micronized 200 g
Corn Starch 20 g
Talc 20 g
Magnesium stearate 2 g
The active ingredient, finely divided by means of an air micronizer, is
added to the other finely powdered ingredients, mixed thoroughly and then
encapsulated in the usual manner.
The foregoing capsules are useful for treating a neoplastic disease by
the oral administration of one or two capsules one to four times a day.
Using the procedure above, capsules are similarly prepared containing
an active ingredient in 50, 250 and 500 mg amounts by substituting 50 g, 250 g
and 500 g of an active ingredient for the 200 g used above.
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COMPOSITION "B"
Soft Gelatin Capsules
One-piece soft gelatin capsules for oral use, each containing 200 mg of
an active ingredient, finely divided by means of an air micronizer, are
prepared
by first suspending the compound in 0.5 ml of corn oil to render the material
capsulatable and then encapsulating in the above manner.
The foregoing capsules are useful for treating a neoplastic disease by
the oral administration of one or two capsules one to four times a day.
COMPOSITION "C"
Tablets
One thousand tablets, each containing 200 mg of an active ingredient,
are prepared from the following types and amounts of ingredients:
Active ingredient, micronized 200 g
Lactose 300 g
Corn starch 50 g
Magnesium stearate 4 g
Light liquid petrolatum 5 g
The active ingredient, finely divided by means of an air micronizer, is
added to the other ingredients and then thoroughly mixed and slugged. The
slugs are broken down by forcing them through a Number Sixteen screen. The
resulting granules are then compressed into tablets, each tablet containing
200
mg of the active ingredient.
The foregoing tablets are useful for treating a neoplastic disease by the
oral administration of one or two tablets one to four times a day.
Using the procedure above, tablets are similarly prepared containing an
active ingredient in 250 mg and 100 mg amounts by substituting 250 g and 100
g of an active ingredient for the 200 g used above.
COMPOSITION "D"
Oral Suspension
One liter of an aqueous suspension for oral use, containing in each
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teaspoonful (5 ml) dose, 50 mg of an active ingredient, is prepared from the
following types and amounts of ingredients:
Active ingredient, micronized 10 g
Citric acid 2 g
Benzoic acid 1 g
Sucrose 790 g
Tragacanth 5 g
Lemon Oil 2 g
Deionized water, q. s. 1000 ml
The citric acid, benzoic acid, sucrose, tragacanth and lemon oil are
dispersed in sufficient water to make 850 ml of suspension. The active
ingredient, finely divided by means of an air micronizer, is stirred into the
syrup
unit uniformly distributed. Sufficient water is added to make 1000 ml.
The composition so prepared is useful for treating a neoplastic disease
at a dose of 1 teaspoonful (15 ml) three times a day.
COMPOSITION "E"
Parenteral Product
A sterile aqueous suspension for parenteral injection, containing 30 mg
of an active ingredient in each milliliter for treating a neoplastic disease,
is
prepared from the following types and amounts of ingredients:
Active ingredient, micronized 30 g
POLYSORBATE 80 5 g
Methylparaben 2.5 g
Propylparaben 0.17 g
Water for injection, q.s. 1000 ml.
All the ingredients, except the active ingredient, are dissolved in the
water and the solution sterilized by filtration. To the sterile solution is
added
the sterilized active ingredient, finely divided by means of an air
micronizer, and
the final suspension is filled into sterile vials and the vials sealed.
The composition so prepared is useful for treating a neoplastic disease
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at a dose of 1 milliliter (lml) three times a day.
COMPOSITION "F"
Suppository, Rectal and Vaginal
One thousand suppositories, each weighing 2.5 g and containing 200
mg of an active ingredient are prepared from the following types and amounts
of ingredients:
Active ingredient, micronized 15 g
Propylene glycol 150 g
Polyethylene glycol #4000, q. s. 2,500 g
The active ingredient is finely divided by means of an air micronizer and
added to the propylene glycol and the mixture passed through a colloid mill
until uniformly dispersed. The polyethylene glycol is melted and the propylene
glycol dispersion is added slowly with stirring. The suspension is poured into
unchilled molds at 40 C. The composition is allowed to cool and solidify and
then removed from the mold and each suppository foil wrapped.
The foregoing suppositories are inserted rectally or vaginally for
treating a neoplastic disease.
COMPOSITION "G"
Intranasal Suspension
One liter of a sterile aqueous suspension for intranasal instillation,
containing 20 mg of an active ingredient in each milliliter, is prepared from
the
following types and amounts of ingredients:
Active ingredient, micronized 15 g
POLYSORBATE 80 5g
Methylparaben 2.5 g
Propylparaben 0.17 g
Deionized water, q. s. 1000 ml.
All the ingredients, except the active ingredient, are dissolved in the
water and the solution sterilized by filtration. To the sterile solution is
added
the sterilized active ingredient, finely divided by means of an air
micronizer, and
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the final suspension is aseptically filled into sterile containers.
The composition so prepared is useful for treating a neoplastic disease,
by intranasal instillation of 0.2 to 0.5 ml given one to four times per day.
An active ingredient can also be present in the undiluted pure form for
use locally about the cutis, intranasally, pharyngolaryngeally, bronchially,
or
orally.
COMPOSITION "H"
Powder
Five grams of an active ingredient in bulk form is finely divided by
means of an air micronizer. The micronized powder is placed in a shaker-type
container.
The foregoing composition is useful for treating a neoplastic disease, at
localized sites by applying a powder one to four times per day.
COMPOSITION "I"
Oral Powder
One hundred grams of an active ingredient in bulk form is finely divided
by means of an air micronizer. The micronized powder is divided into
individual doses of 200 mg and packaged.
The foregoing powders are useful for treating a neoplastic disease, by
the oral administration of one or two powders suspended in a glass of water,
one to four times per day.
COMPOSITION "J"
Insufflation
One hundred grams of an active ingredient in bulk form is finely divided
by means of an air micronizer.
The foregoing composition is useful for treating a neoplastic disease, by
the inhalation of 300 mg one to four times a day.
From the foregoing, it becomes readily apparent that a new and useful
antineoplastic factor and new and useful antineoplastic preparations have been
herein described and illustrated which fulfill all of the aforestated
objectives in a
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remarkably unexpected fashion. It is of course understood that such
modifications, alterations and adaptations as will readily occur to the
artisan
confronted with this disclosure are intended within the spirit of the present
invention.
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